Establishing Experimental Metastases Mouse Model: Implanting GFP Expressing CRC Organoid Cells in PDX Model to Detect Micrometastases

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Generation of Metastases by GFP-labeled CRC Organoids in Recipient Mice

1. To establish an experimental metastasis model of CRC PDXs:
   1. Prepare a CRC organoid cell suspension including 4 x 10⁴ cells in 50 µL of PBS per mouse. Maintain the suspension on ice before use. Use a hemocytometer for counting the cancer cells. (Count the number of tumor cells including single cells and multicellular clusters in the cell suspension).
   2. Anesthetize a mouse with isoflurane inhalation using the small animal inhalation anesthesia device and disinfect the skin with 70% ethanol. Assure the normal rate and depth of respiration and the absence of a toe pinch reflex for proper anesthetization. Vet ointment on the eyes is an option, to prevent ocular dryness while under anesthesia.
   3. Place the anesthetized NOG mouse in a prone position on the laboratory bench. Make a small incision on the lower part of the left flank and then cut the peritoneum to carefully expose the spleen using sterile scissors and tweezers. Grasp the fat tissue attached to the spleen with sterile tweezers, then slowly inject 50 µL of the cell suspension (prepared as above, step 1.1) into the spleen. Be certain that the cell suspension is injected appropriately without outward leakage, from the spleen. Routinely, 4 mice per group are used to evaluate the results.
2. Place all mice on a warm pad after surgery and maintain in a sternal recumbent position until sufficient consciousness is restored. Once the mice have fully recovered and are able to sit up on all four legs , return them to their home cages. To prevent predation, do not allow breeding with non-operated animals in the same cage.
3. Check for suture leakage and confirm that the mice are alive, in the surgically treated group, the next day. Check the mice for signs of illness and measure tumor sizes, employing calipers, in all mice once a week.
4. Observe mice to detect spontaneous metastasis (for periods of up to 2.5 months) and experimental metastasis (1 month) after injection.
5. Sacrifice the mice via anesthesia and cervical dislocation on the indicated days. Dissect primary tumors and various tissues including the liver and lungs. Place the dissected tumors and organs in 5 mL of ice-cold PBS on a 10 cm Petri dish and store them on ice.
6. To detect GFP-positive CRC organoid cells, observe the dissected tissues under a stereo-fluorescence microscope. Acquire images with a CCD camera and prepare using standard image-processing software